October 19, 2013

Plug And Play Synthetic Biology – Rewriting An Entire Genome

A team of scientists from Harvard and Yale have recorded the entire genome of the bacteria E. coli, and in a dramatic demonstration of the potential of rewriting an organism's genetic code, they have improved the bacterium's ability to resist viruses.

"This is the first time the genetic code has been fundamentally changed," according to Farren Isaacs, assistant professor of molecular, cellular, and developmental biology at Yale. "Creating an organism with a new genetic code has allowed us to expand the scope of biological function in a number of powerful ways."

Creating this genomically recoded organism raises the possibility that future researchers might be able to retool nature and create potent new proteins to accomplish a wide variety of purposes — from combating disease to generating new classes of materials. The findings from this groundbreaking study, which changes the rules of biology, were published in Science.

Isaacs and co-author George Church of Harvard Medical School led this research, which is a product of years of studies in the emerging field of synthetic biology, which seeks to re-design natural biological systems for useful purposes.

Encoded by DNA's instructional manual and made up of 20 amino acids, proteins carry out various important functional roles in the cell. A full set of 64 triplet combinations of the four nucleic acids that comprise the backbone of DNA encode amino acids. Triplets are sets of three nucleotides, called codons, and they are the genetic alphabet of life.

For this study, the research team examined the possibility of expanding upon nature's handywork by substituting different codons or letters throughout the genome and then reintroducing entirely new letters to create amino acids not found in nature. This landmark study represents the first time that the genetic code has been completely changed across an organism's genome.

The research team first swapped all 321 instances of a specific codon, or "genetic three-letter word," in E. coli for a supposedly identical word. Then they recoded the original word with a new meaning and new amino acid to eliminate its natural stop sign that terminates protein production. This novel genome allowed the bacteria to resist viral infection by limiting the production of natural proteins that viruses use to infect cells. They then converted the "stop" codon into one that encodes new amino acids, inserted it into the genome in a sort of "plug and play" fashion.

The results set the stage for using the recoded E. coli as a living foundry, capable of biomanufacturing new classes of "exotic" proteins and polymers. The recoded molecules could be the foundation for a new generation of materials, nanostructures, therapeutics, and drug delivery vehicles, Isaacs said.

"Since the genetic code is universal, it raises the prospect of recoding genomes of other organisms," Isaacs said. "This has tremendous implications in the biotechnology industry and could open entirely new avenues of research and applications."

EXPANDING DNA VOCABULARY

The researchers didn't stop there. In a second, related study, they removed every occurrence of 13 different codons across 42 separate E. coli genes, using a different organism for each gene, and replaced them with other codons of the same function. After the replacement, 24 percent of the DNA across the 42 targeted genes had been changed, yet the proteins the genes produced remained identical to those produced by the original genes.

"The first project is saying that we can take one codon, completely remove it from the genome, then successfully reassign its function," said Marc Lajoie, a Harvard Medical School graduate student in the lab of George Church. "For the second project we asked, 'OK, we've changed this one codon, how many others can we change?'"

They found that all 13 codons chosen for the study could be changed. The results of this study also appear in Science.

"That leaves open the possibility that we could potentially replace any or all of those 13 codons throughout the entire genome," Lajoie said.

In the biotech industry, viruses limit productivity. Recoded genomes can confer protection against such viruses and help prevent the spread of potentially dangerous genetically engineered traits to wild organisms.

"In science we talk a lot about the 'what' and the 'how' of things, but in this case, the 'why' is very important," Church said. Church explained how this project is part of an ongoing effort to improve the safety, productivity and flexibility of biotechnology.

"These results might also open a whole new chemical toolbox for biotech production," said Isaacs. "For example, adding durable polymers to a therapeutic molecule could allow it to function longer in the human bloodstream."

To have such a wide-sweeping impact, they needed the ability to change large swaths of the genome all at once.

"If we make a few changes that make the microbe a little more resistant to a virus, the virus is going to compensate. It becomes a back and forth battle," Church said. "But if we take the microbe offline and make a whole bunch of changes, when we bring it back and show it to the virus, the virus is going to say 'I give up.' No amount of diversity in any reasonable natural virus population is going to be enough to compensate for this wildly new genome."

The first study revealed that a genomically recoded organism showed increased resistance to viral infection with just one codon removed. Church said that the same potential "wildly new genome" could prevent engineered genes from escaping into wild populations by making them incompatible with natural genomes. For example, scientists would find this of considerable benefit with strains engineered for drug or pesticide resistance. Incorporating rare, non-standard amino acids could also be used to ensure strains only survive in a laboratory environment.

The research team said that managing a series of hundreds of specific changes was daunting, since a single genetic flaw can spell death for an organism.

"We wanted to develop the ability to efficiently build the desired genome and to very quickly identify any problems—from design flaws or from undesired mutations — and develop workarounds," Lajoie said.

Many novel technologies, developed in the Church lab and the Wyss Institute and with partners in academia and industry, were used in this study. These included next-generation sequencing tools, DNA synthesis on a chip, and MAGE and CAGE genome editing tools. But one of the most important tools they used was the power of natural selection, the researchers added.

"When an engineering team designs a new cellphone, it's a huge investment of time and money. They really want that cell phone to work," Church said. "With E. coli we can make a few billion prototypes with many different genomes, and let the best strain win. That's the awesome power of evolution."